Research

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26 pages, 3406 KiB

Open AccessArticle

Membranolytic Mechanism of Amphiphilic Antimicrobial β-Stranded [KL]_n Peptides

by Fabian Schweigardt, Erik Strandberg, Parvesh Wadhwani, Johannes Reichert, Jochen Bürck, Haroldo L. P. Cravo, Luisa Burger and Anne S. Ulrich

Biomedicines 2022, 10(9), 2071; https://doi.org/10.3390/biomedicines10092071 - 24 Aug 2022

Cited by 1 | Viewed by 1522

Abstract

Amphipathic peptides can act as antibiotics due to membrane permeabilization. KL peptides with the repetitive sequence [Lys-Leu]_n-NH₂ form amphipathic β-strands in the presence of lipid bilayers. As they are known to kill bacteria in a peculiar length-dependent manner, we suggest [...] Read more.

Amphipathic peptides can act as antibiotics due to membrane permeabilization. KL peptides with the repetitive sequence [Lys-Leu]_n-NH₂ form amphipathic β-strands in the presence of lipid bilayers. As they are known to kill bacteria in a peculiar length-dependent manner, we suggest here several different functional models, all of which seem plausible, including a carpet mechanism, a β-barrel pore, a toroidal wormhole, and a β-helix. To resolve their genuine mechanism, the activity of KL peptides with lengths from 6–26 amino acids (plus some inverted LK analogues) was systematically tested against bacteria and erythrocytes. Vesicle leakage assays served to correlate bilayer thickness and peptide length and to examine the role of membrane curvature and putative pore diameter. KL peptides with 10–12 amino acids showed the best therapeutic potential, i.e., high antimicrobial activity and low hemolytic side effects. Mechanistically, this particular window of an optimum β-strand length around 4 nm (11 amino acids × 3.7 Å) would match the typical thickness of a lipid bilayer, implying the formation of a transmembrane pore. Solid-state ¹⁵N- and ¹⁹F-NMR structure analysis, however, showed that the KL backbone lies flat on the membrane surface under all conditions. We can thus refute any of the pore models and conclude that the KL peptides rather disrupt membranes by a carpet mechanism. The intriguing length-dependent optimum in activity can be fully explained by two counteracting effects, i.e., membrane binding versus amyloid formation. Very short KL peptides are inactive, because they are unable to bind to the lipid bilayer as flexible β-strands, whereas very long peptides are inactive due to vigorous pre-aggregation into β-sheets in solution. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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18 pages, 4698 KiB

Open AccessArticle

Antibacterial and Anti-Inflammatory Effects of Apolipoprotein E

by Manoj Puthia, Jan K. Marzinek, Ganna Petruk, Gizem Ertürk Bergdahl, Peter J. Bond and Jitka Petrlova

Biomedicines 2022, 10(6), 1430; https://doi.org/10.3390/biomedicines10061430 - 17 Jun 2022

Cited by 8 | Viewed by 2160

Abstract

Apolipoprotein E (APOE) is a lipid-transport protein that functions as a key mediator of lipid transport and cholesterol metabolism. Recent studies have shown that peptides derived from human APOE display anti-inflammatory and antimicrobial effects. Here, we applied in vitro assays and fluorescent microscopy [...] Read more.

Apolipoprotein E (APOE) is a lipid-transport protein that functions as a key mediator of lipid transport and cholesterol metabolism. Recent studies have shown that peptides derived from human APOE display anti-inflammatory and antimicrobial effects. Here, we applied in vitro assays and fluorescent microscopy to investigate the anti-bacterial effects of full-length APOE. The interaction of APOE with endotoxins from Escherichia coli was explored using surface plasmon resonance, binding assays, transmission electron microscopy and all-atom molecular dynamics (MD) simulations. We also studied the immunomodulatory activity of APOE using in vitro cell assays and an in vivo mouse model in combination with advanced imaging techniques. We observed that APOE exhibits anti-bacterial activity against several Gram-negative bacterial strains of Pseudomonas aeruginosa and Escherichia coli. In addition, we showed that APOE exhibits a significant binding affinity for lipopolysaccharide (LPS) and lipid A as well as heparin. MD simulations identified the low-density lipoprotein receptor (LDLR) binding region in helix 4 of APOE as a primary binding site for these molecules via electrostatic interactions. Together, our data suggest that APOE may have an important role in controlling inflammation during Gram-negative bacterial infection. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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14 pages, 2514 KiB

Open AccessArticle

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli

by Chih-Lung Wu, Ya-Han Chih, Hsin-Ying Hsieh, Kuang-Li Peng, Yi-Zong Lee, Bak-Sau Yip, Shih-Che Sue and Jya-Wei Cheng

Biomedicines 2022, 10(6), 1351; https://doi.org/10.3390/biomedicines10061351 - 08 Jun 2022

Cited by 2 | Viewed by 2351

Abstract

Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34–39 amino acids with an N-terminal [...] Read more.

Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34–39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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18 pages, 2907 KiB

Open AccessArticle

Novel Retro-Inverso Peptide Antibiotic Efficiently Released by a Responsive Hydrogel-Based System

by Angela Cesaro, Rosa Gaglione, Marco Chino, Maria De Luca, Rocco Di Girolamo, Angelina Lombardi, Rosanna Filosa and Angela Arciello

Biomedicines 2022, 10(6), 1301; https://doi.org/10.3390/biomedicines10061301 - 02 Jun 2022

Cited by 5 | Viewed by 1884

Abstract

Topical antimicrobial treatments are often ineffective on recalcitrant and resistant skin infections. This necessitates the design of antimicrobials that are less susceptible to resistance mechanisms, as well as the development of appropriate delivery systems. These two issues represent a great challenge for researchers [...] Read more.

Topical antimicrobial treatments are often ineffective on recalcitrant and resistant skin infections. This necessitates the design of antimicrobials that are less susceptible to resistance mechanisms, as well as the development of appropriate delivery systems. These two issues represent a great challenge for researchers in pharmaceutical and drug discovery fields. Here, we defined the therapeutic properties of a novel peptidomimetic inspired by an antimicrobial sequence encrypted in human apolipoprotein B. The peptidomimetic was found to exhibit antimicrobial and anti-biofilm properties at concentration values ranging from 2.5 to 20 µmol L⁻¹, to be biocompatible toward human skin cell lines, and to protect human keratinocytes from bacterial infections being able to induce a reduction of bacterial units by two or even four orders of magnitude with respect to untreated samples. Based on these promising results, a hyaluronic-acid-based hydrogel was devised to encapsulate and to specifically deliver the selected antimicrobial agent to the site of infection. The developed hydrogel-based system represents a promising, effective therapeutic option by combining the mechanical properties of the hyaluronic acid polymer with the anti-infective activity of the antimicrobial peptidomimetic, thus opening novel perspectives in the treatment of skin infections. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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15 pages, 3386 KiB

Open AccessArticle

Strategy to Enhance Anticancer Activity and Induced Immunogenic Cell Death of Antimicrobial Peptides by Using Non-Nature Amino Acid Substitutions

by Yu-Huan Cheah, Chun-Yu Liu, Bak-Sau Yip, Chih-Lung Wu, Kuang-Li Peng and Jya-Wei Cheng

Biomedicines 2022, 10(5), 1097; https://doi.org/10.3390/biomedicines10051097 - 09 May 2022

Cited by 4 | Viewed by 2081

Abstract

There is an urgent and imminent need to develop new agents to fight against cancer. In addition to the antimicrobial and anti-inflammatory activities, many antimicrobial peptides can bind to and lyse cancer cells. P-113, a 12-amino acid clinically active histatin-rich peptide, was found [...] Read more.

There is an urgent and imminent need to develop new agents to fight against cancer. In addition to the antimicrobial and anti-inflammatory activities, many antimicrobial peptides can bind to and lyse cancer cells. P-113, a 12-amino acid clinically active histatin-rich peptide, was found to possess anti-Candida activities but showed poor anticancer activity. Herein, anticancer activities and induced immunogenic cancer cell death of phenylalanine-(Phe-P-113), β-naphthylalanine-(Nal-P-113), β-diphenylalanine-(Dip-P-113), and β-(4,4′-biphenyl)alanine-(Bip-P-113) substituted P-113 were studied. Among these peptides, Nal-P-113 demonstrated the best anticancer activity and caused cancer cells to release potent danger-associated molecular patterns (DAMPs), such as reactive oxygen species (ROS), cytochrome c, ATP, and high-mobility group box 1 (HMGB1). These results could help in developing antimicrobial peptides with better anticancer activity and induced immunogenic cell death in therapeutic applications. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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19 pages, 3426 KiB

Open AccessArticle

Effect and Mechanisms of Antibacterial Peptide Fraction from Mucus of C. aspersum against Escherichia coli NBIMCC 8785

by Yana Topalova, Mihaela Belouhova, Lyudmila Velkova, Aleksandar Dolashki, Nellie Zheleva, Elmira Daskalova, Dimitar Kaynarov, Wolfgang Voelter and Pavlina Dolashka

Biomedicines 2022, 10(3), 672; https://doi.org/10.3390/biomedicines10030672 - 14 Mar 2022

Cited by 3 | Viewed by 2357

Abstract

Peptides isolated from the mucus of Cornu aspersum could be prototypes for antibiotics against pathogenic bacteria. Information regarding the mechanisms, effective concentration, and methods of application is an important tool for therapeutic, financial, and ecological regulation and a holistic approach to medical treatment. [...] Read more.

Peptides isolated from the mucus of Cornu aspersum could be prototypes for antibiotics against pathogenic bacteria. Information regarding the mechanisms, effective concentration, and methods of application is an important tool for therapeutic, financial, and ecological regulation and a holistic approach to medical treatment. A peptide fraction with MW < 10 kDa was analyzed by MALDI-TOF-TOF using Autoflex™ III. The strain Escherichia coli NBIMCC 8785 (18 h and 48 h culture) was used. The changes in bacterial structure and metabolic activity were investigated by SEM, fluorescent, and digital image analysis. This peptide fraction had high inhibitory effects in surface and deep inoculations of E. coli of 1990.00 and 136.13 mm²/mgPr/µMol, respectively, in the samples. Thus, it would be effective in the treatment of infections involving bacterial biofilms and homogenous cells. Various deformations of the bacteria and inhibition of its metabolism were discovered and illustrated. The data on the mechanisms of impact of the peptides permitted the formulation of an algorithm for the treatment of infections depending on the phase of their development. The decrease in the therapeutic concentrations will be more sparing to the environment and will lead to a decrease in the cost of the treatment. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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10 pages, 1387 KiB

Open AccessArticle

LL-37 and Double-Stranded RNA Synergistically Upregulate Bronchial Epithelial TLR3 Involving Enhanced Import of Double-Stranded RNA and Downstream TLR3 Signaling

by Sara Bodahl, Samuel Cerps, Lena Uller and Bengt-Olof Nilsson

Biomedicines 2022, 10(2), 492; https://doi.org/10.3390/biomedicines10020492 - 19 Feb 2022

Cited by 1 | Viewed by 2257

Abstract

The human host defense peptide LL-37 influences double-stranded RNA signaling, but this process is not well understood. Here, we investigate synergistic actions of LL-37 and synthetic double-stranded RNA (poly I:C) on toll-like receptor 3 (TLR3) expression and signaling, and examine underlying mechanisms. In [...] Read more.

The human host defense peptide LL-37 influences double-stranded RNA signaling, but this process is not well understood. Here, we investigate synergistic actions of LL-37 and synthetic double-stranded RNA (poly I:C) on toll-like receptor 3 (TLR3) expression and signaling, and examine underlying mechanisms. In bronchial epithelial BEAS-2B cells, LL-37 potentiated poly I:C-induced TLR3 mRNA and protein expression demonstrated by qPCR and Western blot, respectively. Interestingly, these effects were associated with increased uptake of rhodamine-tagged poly I:C visualized by immunocytochemistry. The LL-37/poly I:C-induced upregulation of TLR3 mRNA expression was prevented by the endosomal acidification inhibitor chloroquine, indicating involvement of downstream TLR3 signaling. The glucocorticoid dexamethasone reduced LL-37/poly I:C-induced TLR3 expression on both mRNA and protein levels, and this effect was associated with increased IκBα protein expression, suggesting that dexamethasone acts via attenuation of NF-κB activity. We conclude that LL-37 potentiates poly I:C-induced upregulation of TLR3 through a mechanism that may involve enhanced import of poly I:C and that LL-37/poly I:C-induced TLR3 expression is associated with downstream TLR3 signaling and sensitive to inhibition of NF-κB activity. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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16 pages, 10818 KiB

Open AccessArticle

Host Defense Peptides LL-37 and Lactoferrin Trigger ET Release from Blood-Derived Circulating Monocytes

by Frederic V. Schwäbe, Lotta Happonen, Sofie Ekestubbe and Ariane Neumann

Biomedicines 2022, 10(2), 469; https://doi.org/10.3390/biomedicines10020469 - 17 Feb 2022

Cited by 2 | Viewed by 2073

Abstract

Neutrophils are commonly regarded as the first line of immune response during infection or in tissue injury-induced inflammation. The rapid influx of these cells results in the release of host defense proteins (HDPs) or formation of neutrophil extracellular traps (NETs). As a second [...] Read more.

Neutrophils are commonly regarded as the first line of immune response during infection or in tissue injury-induced inflammation. The rapid influx of these cells results in the release of host defense proteins (HDPs) or formation of neutrophil extracellular traps (NETs). As a second wave during inflammation or infection, circulating monocytes arrive at the site. Earlier studies showed that HDPs LL-37 and Lactoferrin (LTF) activate monocytes while neutrophil elastase facilitates the formation of extracellular traps (ETs) in monocytes. However, the knowledge about the impact of HDPs on monocytes remains sparse. In the present study, we investigated the effect of LL-37 and LTF on blood-derived CD14⁺ monocytes. Both HDPs triggered a significant release of TNFα, nucleosomes, and monocyte ETs. Microscopic analysis indicated that ET formation by LL-37 depends on storage-operated calcium entry (SOCE), mitogen-activated protein kinase (MAPK), and ERK1/2, whereas the LTF-mediated ET release is not affected by any of the here used inhibitors. Quantitative proteomics mass spectrometry analysis of the neutrophil granular content (NGC) revealed a high abundance of Lactoferrin. The stimulation of CD14⁺ monocytes with NGC resulted in a significant secretion of TNFα and nucleosomes, and the formation of monocyte ETs. The findings of this study provide new insight into the complex interaction of HDPs, neutrophils, and monocytes during inflammation. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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20 pages, 14463 KiB

Open AccessArticle

In Vivo Evaluation of ECP Peptide Analogues for the Treatment of Acinetobacter baumannii Infection

by Jiarui Li, Guillem Prats-Ejarque, Marc Torrent, David Andreu, Klaus Brandenburg, Pablo Fernández-Millán and Ester Boix

Biomedicines 2022, 10(2), 386; https://doi.org/10.3390/biomedicines10020386 - 05 Feb 2022

Cited by 2 | Viewed by 2035

Abstract

Antimicrobial peptides (AMPs) are alternative therapeutics to traditional antibiotics against bacterial resistance. Our previous work identified an antimicrobial region at the N-terminus of the eosinophil cationic protein (ECP). Following structure-based analysis, a 30mer peptide (ECPep-L) was designed that combines antimicrobial action against Gram-negative [...] Read more.

Antimicrobial peptides (AMPs) are alternative therapeutics to traditional antibiotics against bacterial resistance. Our previous work identified an antimicrobial region at the N-terminus of the eosinophil cationic protein (ECP). Following structure-based analysis, a 30mer peptide (ECPep-L) was designed that combines antimicrobial action against Gram-negative species with lipopolysaccharides (LPS) binding and endotoxin-neutralization activities. Next, analogues that contain non-natural amino acids were designed to increase serum stability. Here, two analogues were selected for in vivo assays: the all-D version (ECPep-D) and the Arg to Orn version that incorporates a D-amino acid at position 2 (ECPep-2D-Orn). The peptide analogues retained high LPS-binding and anti-endotoxin activities. The peptides efficacy was tested in a murine acute infection model of Acinetobacter baumannii. Results highlighted a survival rate above 70% following a 3-day supervision with a single administration of ECPep-D. Moreover, in both ECPep-D and ECPep-2D-Orn peptide-treated groups, clinical symptoms improved significantly and the tissue infection was reduced to equivalent levels to mice treated with colistin, used as a last resort in the clinics. Moreover, treatment drastically reduced serum levels of TNF-α inflammation marker within the first 8 h. The present results support ECP-derived peptides as alternative candidates for the treatment of acute infections caused by Gram-negative bacteria. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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Review

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26 pages, 6671 KiB

Open AccessReview

Chemical Barrier Proteins in Human Body Fluids

by Gergő Kalló, Ajneesh Kumar, József Tőzsér and Éva Csősz

Biomedicines 2022, 10(7), 1472; https://doi.org/10.3390/biomedicines10071472 - 22 Jun 2022

Cited by 2 | Viewed by 2594

Abstract

Chemical barriers are composed of those sites of the human body where potential pathogens can contact the host cells. A chemical barrier is made up by different proteins that are part of the antimicrobial and immunomodulatory protein/peptide (AMP) family. Proteins of the AMP [...] Read more.

Chemical barriers are composed of those sites of the human body where potential pathogens can contact the host cells. A chemical barrier is made up by different proteins that are part of the antimicrobial and immunomodulatory protein/peptide (AMP) family. Proteins of the AMP family exert antibacterial, antiviral, and/or antifungal activity and can modulate the immune system. Besides these proteins, a wide range of proteases and protease inhibitors can also be found in the chemical barriers maintaining a proteolytic balance in the host and/or the pathogens. In this review, we aimed to identify the chemical barrier components in nine human body fluids. The interaction networks of the chemical barrier proteins in each examined body fluid were generated as well. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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30 pages, 10006 KiB

Open AccessReview

Cathelicidin LL-37 in Health and Diseases of the Oral Cavity

by Joanna Tokajuk, Piotr Deptuła, Ewelina Piktel, Tamara Daniluk, Sylwia Chmielewska, Tomasz Wollny, Przemysław Wolak, Krzysztof Fiedoruk and Robert Bucki

Biomedicines 2022, 10(5), 1086; https://doi.org/10.3390/biomedicines10051086 - 07 May 2022

Cited by 18 | Viewed by 3244

Abstract

The mechanisms for maintaining oral cavity homeostasis are subject to the constant influence of many environmental factors, including various chemicals and microorganisms. Most of them act directly on the oral mucosa, which is the mechanical and immune barrier of the oral cavity, and [...] Read more.

The mechanisms for maintaining oral cavity homeostasis are subject to the constant influence of many environmental factors, including various chemicals and microorganisms. Most of them act directly on the oral mucosa, which is the mechanical and immune barrier of the oral cavity, and such interaction might lead to the development of various oral pathologies and systemic diseases. Two important players in maintaining oral health or developing oral pathology are the oral microbiota and various immune molecules that are involved in controlling its quantitative and qualitative composition. The LL-37 peptide is an important molecule that upon release from human cathelicidin (hCAP-18) can directly perform antimicrobial action after insertion into surface structures of microorganisms and immunomodulatory function as an agonist of different cell membrane receptors. Oral LL-37 expression is an important factor in oral homeostasis that maintains the physiological microbiota but is also involved in the development of oral dysbiosis, infectious diseases (including viral, bacterial, and fungal infections), autoimmune diseases, and oral carcinomas. This peptide has also been proposed as a marker of inflammation severity and treatment outcome. Full article

(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)

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